C.25 [substituted(2-propenyl)]milbemycins

ABSTRACT

Antiparasitic milbemycin derivatives having at the C.25 position a substituted 2-propenyl group --C(CH 3 )═CH--R 2  wherein R 2  is a C 3  -C 8  alkyl, alkenyl or alkynyl group which may optionally contain an oxygen or sulphur atom as part of the chain, or a C 3  -C 8  cycloalkyl or cycloalkenyl group, or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may optionally be substituted by one or more alkyl groups or halogen atoms; and process for their preparation.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of application Ser. No. 227,921, filedAug. 3, 1988, now abandoned.

This invention relates to antiparasitic agents and in particular tocompounds related to the milbemycins but having a novel substituent atthe 25-position and to a process for their preparation.

The milbemycins are a group of broad spectrum antiparasitic agentsrelated to the avermectins but differing from them in lacking the sugarresidues at the 13-position. This invention relates specifically to thesubgroup of milbemycins which are characterised by the unsaturationpresent in the 25-position substituent produced by fermenting strains ofthe microorganisms Streptomyces cyaneogriseus ssp noncyanogenus NRRL15773 or Streptomyces thermoarchaensis NCIB 12015. The morphological andcultural properties of the strains NRRL 15773 and NCIB 12015 aredescribed in detail in the European Patent Application No. 0170006 andUK Patent Application GB No. 2 166 436A respectively. The first of thesepatent applications describes the isolation and characterisation ofthirteen individual milbemycins designated LL-F28249α-ν and the seconddescribes a complex of six components designated S541 Factors A-F.

According to the specification of our European patent application No.0214731, published Mar. 18, 1987, we describe a process for preparingnovel avermectins having a modified group at the 25-position, by addinga carboxylic acid or derivative thereof to a fermentation of anavermectin producing organism.

We have now discovered that by adding certain specified carboxylicacids, or derivatives thereof, to a fermentation of the above milbemycinproducing organisms it is possible to obtain novel compounds, related tothe complexes LL-F28249 and S541, but having an unnatural substituentgroup at the 25-position.

Surprisingly, however, the group is not directly attached to the25-position, as in the avermectins, but is linked via a 2-propenylgroup.

The novel compounds are highly active antiparasitic agents havingparticular utility as anthelmintics, ectoparasiticides, insecticides andacaricides.

Conventional chemical transformation reactions can be used to preparefurther derivatives from these compounds. Thus, according to theinvention there are provided compounds having the formula (I): ##STR1##wherein either R is hydrogen and R¹ is hydroxy or R and R¹ are takentogether and are oxo;

R² is a C₃ -C₈ straight or branched-chain alkyl, alkenyl or alkynylgroup which may optionally contain an oxygen or sulphur atom as part ofthe chain, or a C₃ -C₈ cycloalkyl or cycloalkenyl group, or a 3 to 6membered oxygen or sulphur containing heterocyclic ring which may besaturated or fully or partially unsaturated and which may optionally besubstituted by one or more C₁ -C₄ alkyl groups or halogen atoms;

R³ is methyl or ethyl;

R⁴ is hydrogen or methyl; either R⁵ is hydrogen or hydroxy and R⁶ ismethyl or hydroxymethyl, or the two groups R⁵ and R⁶ are taken togetherand are --O--CH₂ --;

R⁷ is hydrogen, methyl or ethyl; and

R⁸ is hydrogen or halogen; with the proviso that when R² is branchedchain alkyl it is not isopropyl.

In the above definition halogen means fluorine, chlorine, bromine oriodine.

A preferred class of compounds have the formula (II): ##STR2## whereinR² and R⁴ are as previously defined.

Particularly preferred are compounds of the formula (II) wherein R² is(1-methylthio)ethyl, 2-pent-4-enyl or 2-butyl.

In accordance with the invention, compounds of the formula (I) whereinR⁸ is hydrogen are prepared by fermenting a strain of the milbemycinproducing organism Streptomyces cyaneogriseus subsp. noncyanogenus NRRL15773 or Streptomyces thermoarchaensis NCIB 12015 in the presence of theappropriate carboxylic acid of the formula R² CO₂ H, wherein R² is aspreviously defined, or a salt, ester or amide thereof, or oxidativeprecursor therefor.

The acid is added to the fermentation either at the time of inoculationor at intervals during the fermentation. Production of the compounds offormula (I) may be monitored by removing samples from the fermentation,extracting with an organic solvent and following the appearance of thecompound of formula (I) by chromatography, for example using highpressure liquid chromatography. Incubation is continued until the yieldof the compound of formula (I) has been maximised, generally for aperiod of from 4 to 6 days.

A preferred level of each addition of the carboxylic acid or derivativethereof is between 0.05 and 10 grams per liter. The best yields of thecompounds of formula (I) are obtained by gradually adding the acid tothe fermentation, for example by daily additions of the acid orderivative thereof over a period of several days. The acid is preferablyadded as a salt, such as the sodium or ammonium salt, but may be addedas an ester, such as the methyl or ethyl ester or as an amide.Alternative substrates which may be used in the fermentation arederivatives which are oxidative precursors for the carboxylic acids;thus, for example suitable substrates would be aminoacids of the formulaR² CH(NH₂)CO₂ H, glyoxylic acids of the formula R² COCO₂ H, methylaminederivatives of the formula R² CH₂ NH₂, substituted lower alkanoic acidsof the formula R² (CH₂)_(n) CO₂ H wherein n is 2, 4 or 6, methanolderivatives of the formula R² CH₂ OH or aldehydes of the formula R² CHO,wherein R² is as previously defined. The media used for the fermentationmay be a conventional complex media containing assimilable sources ofcarbon, nitrogen and other trace elements.

After fermentation for a period of several days at a temperaturepreferably in the range of from 24° to 33° C., the fermentation broth iscentrifuged or filtered and the mycelial cake is extracted with acetoneor methanol. The solvent extract is concentrated and the desired productis then extracted into a water-immiscible organic solvent, such asmethylene chloride, ethyl acetate, chloroform, butanol or methylisobutyl ketone. The solvent extract is concentrated and the crudeproduct containing the compounds of formula (I) is further purified asnecessary by chromatography, for example using preparative reversephase, high pressure liquid chromatography.

The product is generally obtained as a mixture of the compounds offormula (I) wherein R, R¹, R³, R⁴, R⁵, R⁶ and R⁷ are as previouslydefined and, R⁸ is H; however the proportions of the various componentscan vary depending on the particular organism used, the carboxylic acidemployed and the conditions used.

We have found that a broad range of carboxylic acids as defined by R²CO₂ H may be added to the fermentation to yield milbemycin derivativeshaving a novel substituent at the 25-position. Examples of particularacids which may be employed include the following:

2-methylpent-4-enoic acid

2-methylbutyric acid

2-methylthiopropionic acid.

In one particular and preferred aspect of the invention the fermentationis performed in the presence of 2-methylthiopropionic acid sodium saltto yield predominantly the compounds of formula (II) wherein R² is(1-methylthio)ethyl and R⁴ is H.

In another preferred aspect of the invention the fermentation isperformed in the presence of 2-methylbutyric acid sodium salt to yieldpredominantly the compound of formula (II) wherein R² is 2-butyl and R⁴is H or methyl.

In a further preferred aspect of the invention the fermentation isperformed in the presence of 2-methylpent-4-enoic acid sodium salt toyield predominantly the compound of formula (II) wherein R² is2-pent-4-enyl and R⁴ is H.

Compounds of the formula (I) wherein R⁸ is H may be converted to thecorresponding compounds wherein R⁸ is halogen using publishedprocedures. The reaction is performed by first protecting the hydroxylgroups present, at the 5, 6, 8a and 23 positions, for example as thet-butyldimethylsilyloxy acetyl derivative, followed by halogenation atthe 13 position using for example N-bromosuccinimide in the presence oflight followed by deprotection. Alternatively the process is performedby epoxidation of the 14, 15 double bond using a peracid, for examplem-chloroperbenzoic acid. The hydroxyl groups present at the 5, 6, 8a and23 positions are protected, for example as the t-butyldimethylsilylderivative, and the compound is treated with a pre-formed complex ofhydrazoic acid (HN₃) and triethylaluminium in an inert solvent to givethe 15-hydroxy-Δ¹³,14 -milbemycin derivative. This compound is thenconverted to the 13-halogeno-milbemycin by treatment with an appropriatehalogenating reagent, for example phosphorus tribromide, to give the13-bromo derivative. These steps together with appropriate reagents andreaction conditions are described in the European patent application No.0184989.

The halo-compounds, as well as being active anti-parasitic agents, canserve as intermediates for preparation of the corresponding avermectinderivatives. Thus the protected halo-derivative is converted to the13-acetoxy derivative by treatment with a mixture of sodium acetate inacetic acid. Subsequent reaction with sodium hydroxide gives the13-hydroxy milbemycin to which the L-oleandrosyl-α-L-oleandrosyloxygroup may be attached by reaction with an acetohalo derivative of thedisaccharide. The product is then finally deprotected to give theavermectin. These steps together with appropriate reagents and reactionconditions are described in U.S. Pat. No. 4093629 and British patent No.1579118. Certain of the avermectin products produced by this procedureare novel compounds, not previously obtainable.

The compounds of formula (I) wherein R and R¹ are taken together and areoxo may be isolated from the mixture of products obtained in thefermentation of the organism Streptomyces thermoarchaensis NCIB 12015 orof a mutant thereof as described in British patent application GB No.2176182A, or they may be obtained by oxidation of the correspondingcompounds of formula (I) wherein R is H and R¹ is OH. Appropriatereagents and procedures for performing the oxidation are described in GBNo. 2176182A.

The compounds of formula I wherein R⁴ is H may also be prepared from thecorresponding compounds wherein R⁴ is CH₃ by demethylation. Thisreaction is achieved by treating the 5-methoxy compound, or a suitablyprotected derivative thereof, with mercuric acetate and hydrolysing theresulting 3-acetoxy enol ether with dilute acid to give the 5-ketocompound. This is then reduced using, for example, sodium borohydride toyield the 5-hydroxy derivative. Appropriate reagents and reactionconditions for these steps are described in U.S. Pat. No. 4423209.

The compounds of the invention are highly active antiparasitic agentshaving particular utility as anthelmintics, ectoparasiticides,insecticides and acaricides.

Thus the compounds are effective in treating a variety of conditionscaused by endoparasites including, in particular, helminthiasis which ismost frequently caused by a group of parasitic worms described asnematodes and which can cause severe economic losses in swine, sheep,horses and cattle as well as affecting domestic animals and poultry. Thecompounds are also effective against other nematodes which affectvarious species of animals including, for example, Dirofilaria in dogsand various parasites which can infect humans includinggastro-intestinal parasites such as Ancylostoma, Necator, Ascaris,Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius andparasites which are found in the blood or other tissues and organs suchas filiarial worms and the extra intestinal stages of Strongyloides andTrichinella.

The compounds are also of value in treating ectoparasite infectionsincluding in particular arthropod ectoparasites of animals and birdssuch as ticks, mites, lice, fleas, blowfly, biting insects and migratingdipterous larvae which can affect cattle and horses.

The compounds are also insecticides active against household pests suchas the cockroach, clothes moth, carpet beetle and the housefly as wellas being useful against insect pests of stored grain and of agriculturalplants such as spider mites, aphids, caterpillars, fire ants, termitesand against migratory orthopterans such as locusts.

The compounds of formula (I) are administered as a formulationappropriate to the specific use envisaged and to the particular speciesof host animal being treated and the parasite, or insect involved. Foruse as an anthelmintic the compounds may be administered orally in theform of a capsule, bolus, tablet or preferably a liquid drench, oralternatively, they may be administered by injection or as an implant.Such formulations are prepared in a conventional manner in accordancewith standard veterinary practice. Thus capsules, boluses or tablets maybe prepared by mixing the active ingredient with a suitable finelydivided diluent or carrier additionally containing a disintegratingagent and/or binder such as starch, lactose, talc, magnesium stearateetc. A drench formulation may be prepared by dispersing the activeingredient in an aqueous solution together with dispersing or wettingagents etc. and injectable formulations may be prepared in the form of asterile solution which may contain other substances, for example, enoughsalts or glucose to make the solution isotonic with blood. Theseformulations will vary with regard to the weight of active compounddepending on the species of host animal to be treated, the severity andtype of infection and the body weight of the host. Generally for oraladministration a dose of from about 0.001 to 10 mg per Kg of animal bodyweight given as a single dose or in divided doses for a period of from 1to 5 days will be satisfactory but of course there can be instanceswhere higher or lower dosage ranges are indicated and such are withinthe scope of this invention.

As an alternative the compounds may be administered with the animalfeedstuff and for this purpose a concentrated feed additive or premixmay be prepared for mixing with the normal animal feed.

For use as an insecticide and for treating agricultural pests thecompounds are applied as sprays, dusts, emulsions and the like inaccordance with standard agricultural practice.

For human use the compounds are administered as a pharmaceuticallyacceptable formulation in accordance with standard medical practice.

This invention is illustrated by the following Examples in whichExamples 1-7 are Examples of the preparation of compounds of the formula(I) and Examples 8 and 9 illustrate the anti-parasitic and insecticidalactivity of the compounds. The designations milbemycin A₃ and milbemycinB₂ refer to the natural milbemycins (Antibiotic B-41) of formula (II)but lacking the hydroxyl group at the 23-position and with methyl at the25-position wherein R⁴ is hydrogen or methyl respectively.

EXAMPLE 123-Hydroxy-25-desmethyl-25-(1-methyl-3-methylthio-but-1-enyl)milbemycinA₃ (formula II; R⁴ =H, R² =CH₃ CH(SCH₃)--)

A frozen mycelial preparation of S. cyaneogriseus subsp. noncyanogenusNRRL 15773 was inoculated into 50 mls of medium containing glucose (1g), distillers solubles (0.75 g) and yeast extract (0.25 g) andincubated at 28° C. for 2 days with agitation. This culture was used toinoculate a three liter flask containing 600 mls of the same mediumwhich was incubated as above for a further two days. This culture (100mls.) was used to inoculate 2.5 liters of medium of the followingcomposition, contained in a 5 liter fermenter:

Glucose (68.5 g), Trusoy flour (31.25 g), cane molasses (3.75 g),dipotassium hydrogen phosphate (0.375 g), calcium carbonate (3.125 g),and N-morpholinopropane sulphonic acid (25 g). The fermentation wasincubated at 28° C. with agitation at 1200 rpm and aeration of 2.5liters per minute.

2-Methylthiopropionic acid (1 g) was added after 24 hours incubation andthe fermentation was continued for a further 120 hours. The mycelium wasremoved by filtration and extracted with acetone (1.5 liters) followedby methylene chloride (1.5 liters).

The methylene chloride extracts from six fermentations as describedabove were combined and concentrated to dryness to give the crudeproduct as a dark gum. This material was dissolved in a mixture ofmethylene chloride and hexane (1:4) and added to a column of basicalumina (160 g). The column was eluted with 400 ml of solvent of thesame composition. The column was then eluted with methanol collecting100 ml fractions. Fractions 1-4 were combined and concentrated to yieldpartially purified material (15.8 g) as a yellow oil. This oil waspartitioned between isooctane (100 ml) and methanol (50 ml). Themethanol layer was separated and water (100 ml) added. This mixture wasextracted with methylene chloride (2×50 ml) and the lower layer wasdried with anhydrous sodium sulphate and evaporated to give partiallypurified product as an oil (10 g). This material was dissolved in amixture of methylene chloride and ethyl acetate (3:1) and added to acolumn of silica (250 g). The column was eluted with a solvent of thesame composition and 50 ml fractions were collected. The fractionscontaining the desired product were identified by analytical HPLC,combined and concentrated to give, the semipurified milbemycinderivative. A sample of this material (150 mg) was further purified bychromatography on a C18 Zorbax ODS (Trademark, Dupont) column (21 mm×25cm) eluting with a mixture of methanol and water (78:22) at a flow rateof 9 mls. per minute. Fractions collected at around 70 minutes werecombined and the solvent was evaporated to yield the compound of formula(II) wherein R² is (1-methylthio)ethyl and R⁴ is H, as a white powder,m.p. 120°-122° C. The structure of the product was confirmed by massspectrometry and by C13 nuclear magnetic resonance spectroscopy asfollows:

Fast atom bombardment mass spectrometry was performed on a VG model7070E mass spectrometer using a sample matrix of glycerol, thioglycerol,water and sodium chloride. (M+Na⁺) observed at m/e 667 (theoretical667).

Electron impact mass spectrometry was performed using a VG model 7070Fmass spectrometer. The m/e values for the principal fragments were 644(M⁺), 626, 608, 579, 561, 498, 480, 450, 354, 315, 185, 151.

The C13 nuclear magnetic resonance spectral data were obtained on aNicolet QE 300 spectrometer with a sample concentration of 20 mg/ml indeuterochloroform. The chemical shifts in parts per million relative totetramethylsilane were 11.6, 14.0 (two unresolved signals), 15.6, 19.9,20.8, 22.3, 34.8, 35.9, 36.0, 36.2, 38.4, 40.8, 41.2, 45.8, 48.5, 67.7(two unresolved signals), 68.5, 68.7, 69.1, 76.6, 79.3, 80.3, 99.8,118.0, 120.2, 120.4, 123.4, 132.6, 134.1, 137.4, 138.0, 139.5, 142.8,173.4.

EXAMPLE 2 23-Hydroxy-25-desmethyl-25-(1,3-dimethylpent-1-enyl)milbemycinA₃ (formula (II, R⁴ =H, R² =CH₃ CH₂ CH(CH₃)--)

The conditions of Example 1 were followed except that 2-methylbutyricacid was used as substrate. The methylene chloride extract derived fromtwelve 2.5 liter fermentations was evaporated to give a brown oil (27.2g) which was partially purified by dissolving in a mixture ofdichloromethane and ethyl acetate (4:1) and adding to a column of silica(250 g). The column was eluted with solvent of the same composition and50 ml fractions were collected. The combined fractions 4 and 5 were usedin Example 3 and the fractions 7-11 containing the title compound(identified by thin layer chromatography) were combined and evaporatedto give the semi-purified milbemycin derivative (2.73 g). This materialwas further purified by chromatography on a Dynamax 60-A C-18(Trademark, Rainin) column (41×250 mm) eluting with a mixture of waterand methanol (1:4). Final purification was achieved by chromatography ona C18 Zorbax ODS (Trademark, Dupont) column (21 mm×25 cm) eluting with amixture of water and methanol (1:4) at a flow rate of 9 mls. per minute.Fractions collected at around 90 minutes were combined and the solventwas evaporated to yield the title compound of formula (II) wherein R² is2-butyl and R⁴ is H, as a white powder having a melting point of125°-128° C. The structure of the product was confirmed by massspectrometry and by C13 and proton nuclear magnetic resonancespectroscopy as follows:

Fast atom bombardment mass spectrometry was performed on a VG model7070E mass spectrometer using a sample matrix of glycerol, thioglycerol,water and sodium chloride. (M+Na⁺) observed at m/e 649 (theoretical649).

Electron impact mass spectrometry was performed using a VG model 7070Fmass spectrometer. The m/e values for the principal fragments were 626(M⁺), 608, 590, 480, 462, 425, 354, 314, 248, 151.

The C13 nuclear magnetic resonance spectral data were obtained on aNicolet QE 300 spectrometer with a sample concentration of 20 mg/ml indeuterochloroform. The chemical shifts in parts per million relative totetramethylsilane were 11.5, 12.1, 14.1, 15.7, 20.1, 20.4, 22.4, 30.2,33.8, 34.9, 36.1, 36.2, 36.3, 40.9, 41.3, 45.8, 48.6, 67.8, 67.9, 68.6,68.6, 69.5, 76.8, 79.4, 80.3, 99.9, 118.2, 120.4, 123.5, 131.6, 136.2,137.5, 138.0, 139.5, 142.9, 173.5.

The proton magnetic resonance spectral data were obtained on a NicoletQE 300 spectrometer with the sample in deuterochloroform solution. Thechemical shifts of characteristic signals in parts per million relativeto tetramethylsilane were 5.85-5.7 (m, 2H), 5.45 (bs, 1H), 5.45-5.25 (m,2H), 5.20 (d, 1H), 4.98 (m, 1H), 4.70 (m, 2H), 4.32 (t, 1H), 3.99 (d,1H), 3.90 (s, 1H), 3.80 (d, 1H), 3.61 (d, 1H), 3.30 (m, 1H), 1.90 (bs,3H), 1.64 (s, 3H), 1.56 (s, 3H), 1.03 (d, 3H), 0.84 (d, 3H).

EXAMPLE 3 23-Hydroxy-25-desmethyl-25-(1,3-dimethylpent-1-enyl)milbemycinB₂ (formula II, R⁴ =CH₃, R² =CH₃ CH₂ CH(CH₃)--)

The combined fractions 4 and 5 from Example 2 were evaporated to drynessto give 2.25 g of residue which was further purified in two equalportions by chromatography on a Dynamax 60-A C-18 (Trademark, Rainin)column (41×250 mm) eluting with a mixture of methanol and water (85:15)at a flow rate of 45 mls per minute. Fractions eluting at around 70minutes containing the title compound were combined and the solventevaporated to give the semi-purified milbemycin. This material wasfurther purified by column chromatography on silica gel, eluting with amixture of dichloromethane and ethyl acetate (4:1) and finally purifiedby high pressure liquid chromatography using an Ultrasphere-ODS(Trademark, Beckman) column (10×250 mm) eluting with a mixture ofmethanol and water (72:28) at a flow rate of 4 mls per minute. Fractionseluting at around 70 minutes contained the pure title compound and wereevaporated to dryness. The structure of the product was confirmed byfast atom bombardment mass spectrometry performed on a VG model 7070Emass spectrometer using a sample matrix of trigol, tetragol, water andsodium chloride. (M+Na⁺) observed at m/e 663 (theoretical 663).

EXAMPLE 423-Hydroxy-25-desmethyl-25(1,3-dimethyl-hexa-1,5-dienyl)milbemycin A₃(formula II, R⁴ =H, R² =CH₂ =CHCH₂ CH(CH₃)--)

The conditions of Example 1 were followed except that2-methylpent-4-enoic acid was used as substrate. The crude extractderived from twelve 2.5 liter fermentations (30 g) was partiallypurified by column chromatography using alumina (active, neutral,Brockmann grade 1, BDH Chemicals Ltd., 500 g) eluting initially withhexane and then a mixture of dichloromethane and methanol (1:1),collecting 200-250 ml fractions. The fractions were analysed by t.l.c.and those containing the desired product were combined and evaporatedunder vacuum. A portion of this material (10 g) was further purified bycolumn chromatography using silica gel (Kieselgel 60, 230-400 mesh,Merck, 500 g) eluting with a mixture of dichloromethane and ethylacetate (4:1). Fractions containing the desired product were combinedand evaporated under vacuum. This material was further purified inbatches by HPLC using a Dynamax 60-A C-18 (Trademark, Rainin) column(41×250 mm) eluting with a mixture of methanol and water (4:1) at a flowrate of 52 ml per minute. Fractions were analysed by HPLC using anUltrasphere--ODS (5 μm) (Trademark--Beckman) column (10×250 mm) elutingwith a methanol-water gradient and those fractions containing the titlecompound were combined and evaporated under vacuum. Final purificationwas achieved by chromatography using an Ultrasphere--ODS (5 μm)(Trademark--Beckman) HPLC column (10×250 mm) eluting with a mixture ofwater and methanol (18:82) at a flow rate of 3 ml per minute. Fractionscontaining the pure title compound were combined and evaporated todryness under vacuum. The structure of the product was confirmed by massspectrometry and by proton nuclear magnetic resonance spectroscopy asfollows:

Fast atom bombardment mass spectrometry was performed on a VG model7070E mass spectrometer using a sample matrix of glycerol, thioglycerol,water and sodium chloride. (M+Na⁺) observed at m/e 661 (theoretical661).

Electron impact mass spectrometry was performed using a VG model 7070Fmass spectrometer. The m/e values for the principal fragments were 620(M--H₂ O)⁺, 602 (M--2H₂ O)⁺, 492, 470, 442, 425.

The proton magnetic resonance spectral data were obtained on a NicoletQE 300 spectrometer with the sample in deuterochloroform solution. Thechemical shifts of characteristic signals in parts per million relativeto tetramethylsilane were 5.95-5.70 (3H, m), 5.45 (bs, 1H), 5.4-5.27 (m,2H), 5.23 (d, 1H), 5.10 (d, 1H), 5.05 (bs, 1H), 4.98 (m, 1H), 4.70 (m,2H), 4.31 (t, 1H), 4.00 (d, 1H), 3.9-3.8 (m, 2H), 3.8 (d, 1H), 3.7-3.6(m, 1H), 3.60 (d, 1H), 3.3 (m, 1H), 1.90 (bs, 3H), 1.65 (s, 3H), 1.57(s, 3H), 1.43 (t, 1H), 1.03 (d, 3H), 0.98 (d, 3H), 0.83 (d, 3H).

EXAMPLE 523-Hydroxy-25-desmethyl-25(1,3-dimethyl-hexa-1,5-dienyl)milbemycin A₃(formula II, R⁴ =H, R² =CH₂ =CHCH₂ CH(CH₃)--)

An inoculum of Streptomyces thermoarchaensis NCIB 12015 was prepared bywashing with sterile water (10 ml) a slant culture which was maintainedon a medium prepared from yeast extract (0.5 g), malt extract (30 g),peptone (5 g) and agar (20 g) in tap water (1 liter) adjusted to pH 5.4.This inoculum was transferred to an Erlenmeyer flask containing 50 ml ofa medium containing glucose (0.8 g), glycerol (0.8 g), peptone (0.8 g),NaCl (0.06 g), CaCO₃ (0.02 g) in tap water adjusted to pH 7. The flaskwas incubated at 28° C. on a rotary shaker operating at 170 rpm for 48hours. The resultant vegetative growth was transferred to a 3 litermechanically agitated vessel containing glucose (5 g), dextrin (50 g),Trusoy flour (25 g), cane molasses (3 g), K₂ HPO₄ (0.25 g), CaCO₃ (2.5g) and 3-(N-morpholino)propanesulphonic acid (42.0 g) in tap water (2liters) adjusted to pH 6.5 by addition of 1N NaOH solution. The vesselwas incubated at 28° C. with an agitation speed of 1000 rpm and an airflow of 2 liters per minute.

After 72 hours 2-methylpent-4-enoic acid (0.8 ml) was added and thefermentation mailntained for a further 72 hours. The mycelium wasrecovered by filtration and suspended in acetone (500 ml) for 1 hourbefore filtering. The solid was re-suspended in acetone (500 ml) for 0.5hours and filtered. The combined filtrates were evaporated to drynessunder vacuum to yield a dark coloured oil (8.1 g). This material waspartially purified by column chromatography on silica gel (Kieselgel 60,230-400 mesh, Merck--trademark, 240 g) eluting with a mixture ofdichloromethane and ethyl acetate (4:1), collecting 50 ml fractions.These were analysed by t.l.c. and those fractions containing the titlecompound were combined and evaporated under vacuum. This material wasfurther purified by column chromatography on silica gel (130 g) elutingwith mixtures of dichloromethane and diethyl ether initially 4:1 then2:1 and finally with diethyl ether alone. Fractions rich in the desiredproduct were combined, evaporated to dryness under vacuum and dissolvedin methanol (3 ml). This solution was purified by HPLC using a Dynamax60-A C-18 (Trademark, Rainin) column (41×250 mm) eluting with a mixtureof methanol and water (4:1) at a flow rate of 52 ml per minute.Fractions were collected at 3-5 minute intervals and analysed by HPLC.Those containing the desired product were combined and evaporated undervacuum. Final purification was achieved by HPLC using an Ultrasphere-ODS(5 μm) (Trademark--Beckman) HPLC column (10×250 mm) eluting with amixture of water and methanol (18:82) at a flow rate of 3 ml per minute.Appropriate fractions were combined and evaporated under vacuum to givea white powder identical in all respects to the product of Example 4.

EXAMPLE 6

The procedures of Examples 2 and 3 are repeated except that thesubstrates listed below are used in place of 2-methyl butyric acid. Ineach instance, the corresponding milbemycin A₃ and B₂ derivatives offormula (II) (R⁴ =H and CH₃) are obtained.

    ______________________________________                                        Substrate            R.sup.2                                                  ______________________________________                                        2-methylvaleric acid pent-2-yl                                                1-cyclohexane carboxylic acid                                                                      cyclohexen-1-yl                                          thiopene-2-carboxylic acid                                                                         thien-2-yl                                               3-furoic acid        3-furyl                                                  cyclobutane carboxylic acid                                                                        cyclobutyl                                               cyclopentane carboxylic acid                                                                       cyclopentyl                                              cyclohexane carboxylic acid                                                                        cyclohexyl                                               cycloheptane carboxylic acid                                                                       cycloheptyl                                              3-cyclohexene-1-carboxylic acid                                                                    cyclohex-3-enyl                                          2-methylpent-2-enoic acid                                                                          2-penten-2-yl                                            2-furoic acid        2-furyl                                                  5-methylthiophene-2-carboxylic acid                                                                5-methylthien-2-yl                                       1-methylcyclopropane carboxylic acid                                                               1-methylcyclopropyl                                      cyclopropane carboxylic acid                                                                       cyclopropyl                                              2-methylcyclopropane carboxylic acid                                                               2-methylcyclopropyl                                      2-methyl-4-methoxybutyric acid                                                                     4-methoxybut-2-yl                                        tetrahydrothiophene-3-carboxylic acid                                                              tetrahydrothien-3-yl                                     3-methylcyclobutane carboxylic acid                                                                3-methylcyclobutyl                                       3-methylene cyclobutane carboxylic acid                                                            3-methylenecyclobutyl                                    2-methyl-4-methylthiobutanoic acid                                                                 4-methylthiobut-2-yl                                     tetrahydrothiopyran-4-carboxylic acid                                                              tetrahydrothiopyran-4-yl                                 3-cyclopentenemethanol                                                                             cyclopent-3-enyl                                         ______________________________________                                    

EXAMPLE 7

The procedure of Example 5 is followed except that the followingsubstrates are used in place of 2-methylpent-4-enoic acid to produceformula (II) compounds wherein R⁴ is H and R² is as listed below.

    ______________________________________                                        Substrate            R.sup.2                                                  ______________________________________                                        thiophene-3-carboxylic acid                                                                        thien-3-yl                                               hydroxymethylcyclopentane                                                                          cyclopentyl                                              3-thiophene carboxaldehyde                                                                         thien-3-yl                                               3-cyclohexylpropionic acid                                                                         cyclohexyl                                               3-cyclopentylpropionic acid                                                                        cyclopentyl                                              hydroxymethylcyclobutane                                                                           cyclobutyl                                               3-cyclopentyl-1-propanol                                                                           cyclopentyl                                              cyclobutylmethylamine                                                                              cyclobutyl                                               ethyl cyclobutanecarboxylate                                                                       cyclobutyl                                               2-(cyclobutylcarbonyl)propionic acid                                                               cyclobutyl                                               ethyl 2-(3-thiophenecarbonyl)propionate                                                            thien-3-yl                                               1-methylcyclopropane carboxylic acid                                                               1-methylcyclopropyl                                      2,3-dimethylbutyric  1,2-dimethylpropyl                                       2-methylhexanoic acid                                                                              hex-2-yl                                                 2-cyclopropyl propionic acid                                                                       1-cyclopropylethyl                                       4-methylenecyclohexane carboxylic acid                                                             4-methylenecyclohexyl                                    3-methylcyclohexane carboxylic acid                                                                2-methylcyclohexyl                                       (cis/trans)                                                                   1-cyclopentene carboxylic acid                                                                     cyclopenten-1-yl                                         tetrahydropyran-4-carboxylic acid                                                                  tetrahydropyran-4-yl                                     2-chlorothiophene-4-carboxylic acid                                                                2-chlorothien-4-yl                                       (S)-2-methylpentanoic acid                                                                         (S)-pent-2-yl                                            (R)-2-methylpentanoic acid                                                                         (R)-pent-2-yl                                            ______________________________________                                    

EXAMPLE 8 Anthelmintic Activity

Anthelmintic activity was evaluated against Caenorhabditis elegans usingthe in vitro screening test described by K. G. Simpkin and G. L. Colesin Parasitology, 1979, 79, 19. The product of Examples 1 and 2 killed100% of the worms at a well concentration of 0.01 parts per million.

EXAMPLE 9 Insecticidal Activity

Activity against the larval stage of the blowfly Lucilia cuprina (Qstrain) is demonstrated using a standard procedure in which first instarlarvae are kept in contact with filter paper treated with test compound.The test compound is first applied to the paper as an acetone solution.The treated filter papers are then placed into tubes containing 1 ml ofnewborn calf serum and the first instars are added. The product ofExamples 1 and 2 killed 100% of the larvae when applied to the filterpaper at a level of 10 milligrams per square meter.

We claim:
 1. A compound having the formula: ##STR3## wherein R² is a 3to 6 membered oxygen or sulphur containing heterocyclic ring which maybe saturated or fully or partially unsaturated and which may besubstituted by one or more C₁ -C₄ alkyl groups or halogen atoms;R⁴ ishydrogen or methyl.
 2. A composition for the treatment or prevention ofparasitic infections in humans and animals which comprises a compound ofclaim 1 together with an inert diluent or carrier.
 3. A composition asclaimed in claim 2 in the form of a liquid drench or an oral orinjectable formulation.
 4. A composition as claimed in claim 2 in theform of an animal feedstuff or in the form of a premix or supplement foraddition to animal feed.
 5. A method of combating insect or parasiteinfections or infestations in humans and animals and agricultural orhorticultural pest infestations, which comprises applying an effectiveamount of a compound according to claim 1 to the organism responsiblefor said infection or infestation or to the location thereof.
 6. Acompound according to claim 1 wherein R² is thienyl.
 7. A compoundaccording to claim 6 wherein R² is thien-2-yl.
 8. A compound accordingto claim 1 wherein R² is furyl.
 9. A compound according to claim 8wherein R² is 2-furyl.
 10. A compound according to claim 1 wherein R² istetrahydrothien-3-yl.
 11. A compound according to claim 1 wherein R² istetrahydrothiopyran-4-yl.